Western blot

Western blot is a type of laboratory test used to confirm the diagnosis of certain diseases. It is a form of immunoblotting, which is a kind of test that uses a special protein mixture to identify specific proteins found in the patient’s blood. The test originated in the 1970s and became the standard for confirming the diagnosis of Lyme disease and human immunodeficiency virus (HIV).

Background

A blot test is a scientific procedure that uses a special type of membrane to detect the presence of proteins in a biological sample. The sample is transferred first to a gel and then to the membrane, which helps separate it from the sample and makes it easier to identify. There are multiple types of blot tests, which are used to separate and identify different types of biological materials, including deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and various types of viruses.

The Western blot test got its name as a nod to the Southern blot test, another test that uses a similar technique. The Southern blot test, which is used for research with DNA, was named for Edwin Southern, the British scientist who developed it. Following the development of the Southern blot test, another group of scientists working with American molecular biologist George Stark devised another test for detecting RNA. They jokingly named the test the Northern blot, as a play on the name of the Southern blot technique. When American molecular biologist W. Neal Burnette developed another technique that used immunoblotting in 1979, he and a colleague decided to continue the practice of playing on Southern’s name and called the new test the Western blot.

Western blot testing is used to determine the presence of Lyme disease and HIV infections. Lyme disease is a bacterial illness carried by deer ticks. People get the disease when they are bitten by an infected tick; the bite often causes a telltale bull’s-eye rash. The disease causes system-wide inflammation that leads to headaches, fever, and other flu-like symptoms. It can progress to more serious conditions, such as arthritis and neurological complications. HIV is a virus caused by exposure to the blood, semen, or vaginal fluids of an infected person. It also causes flu-like symptoms that may progress to acquired immunodeficiency syndrome (AIDS).

Overview

The Western blot test is used to detect and analyze proteins in samples containing suspected diseases. It is most commonly used to confirm the diagnosis of Lyme disease and HIV infections that have been identified by other tests. The Western blot test uses blood samples that have been added to a special gel called SDS-polyacrylamide gel (PAGE). This gel is then treated with electrophoresis to separate specific proteins from the sample. Electrophoresis is a technique that uses an electrical current applied to a fluid to separate it into different parts. The proteins are negatively charged, so they move toward the positively charged part of the electrophoresis device, which is called an anode. Electrophoresis also helps to separate the proteins by weight.

Once the proteins are separated, they are transferred to a special membrane. The membrane is usually made of nitrocellulose (NC) or polyvinylidene difluoride (PVDF). This process is called blotting. The special membrane makes it easier to see and identify the proteins that are present. The membrane is chemically treated, so only the proteins of interest will respond to further testing; this process is called blocking. This blocking, which uses substances such as nonfat dry milk, casein, gelatin, or other chemicals, limits the amount of reaction that can happen with proteins other than the ones being studied.

After blocking, antibodies that react to the protein of interest are introduced to the membrane and the sample it contains. These protein-specific antibodies are called primary antibodies and react only with the protein for which they have been designed. Additional antibodies called secondary antibodies are often added to help with the process. They are added after the membrane is washed to remove any of the primary antibodies that did not react with the proteins of interest. A marker solution that helps make the proteins of interest even more visible may be added to help in determining how much of the protein is present. This creates visible bands of the protein that can be analyzed.

The Western blot test is usually used to confirm the results of other tests that have already been performed. The test provides more accurate results than the SDS-PAGE test, or a test that uses electrophoresis and a gel to separate the proteins. It is also more accurate than the enzyme-linked immunosorbent assay, or ELISA test. This test uses antibodies or enzymes to test a sample. The Western blot is better at identifying specific proteins and is used to confirm the diagnosis made with these other methods. It was also used to help rule out false positive results—situations in which another test indicated the presence of the disease when it was not really existent—that are sometimes generated by the SDS-PAGE and ELISA tests.

For several decades, the Western blot was the standard test used to confirm the results of the ELISA test for HIV. However, newer tests were developed that proved to be even more effective than the Western blot. These tests are part of an algorithm approved for use by the US Centers for Disease Control and Prevention (CDC) in 2014. The combination of test results is thought to provide a quicker diagnosis, which is important to getting earlier treatment for those who are HIV positive. This can delay the development of AIDS, resulting in a better outcome for the patient. It can also help prevent the patient from spreading the disease to sexual partners.

Bibliography

Begun, Habeebuynnisa, Periyasamy Murugesan, and Anjana Devi Tangutura. "Western Blotting: A Powerful Staple in Scientific and Biomedical Research." BioTechniques, vol. 73, no. 1, 1 July 2022, pp. 58-69, doi.org/10.2144/btn-2022-0003. Accessed 17 Nov. 2024.

Gingrich, Jane. “What Is the Western Blot Test?” Sciencing, 24 Apr. 2017, sciencing.com/western-blot-test-6046826.html. Accessed 27 Sept. 2018.

“Limitations for the Use of HIV-1 Western Blot in Plasma/Serum.” Association for Public Health Laboratories, June 2015, www.aphl.org/aboutAPHL/publications/Documents/ID‗HIV-1-1-WesternBlotBrief‗62015.pdf. Accessed 27 Sept. 2018.

Mukhopadhyay, Rajendrani. “The Men behind Western Blotting.” ASBMB Today, Mar. 2012, www.asbmb.org/asbmbtoday/asbmbtoday‗article.aspx?id=16084. Accessed 27 Sept. 2018.

Mukhopadhyay, Rajendrani. “W. Neal Burnette: The Man behind the Western Blot.” ASBMB Today, Jan. 2012, www.asbmb.org/asbmbtoday/asbmbtoday‗article.aspx?id=15434. Accessed 27 Sept. 2018.

“Overview of Western Blotting.” ThermoFisher Scientific, www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-western-blotting.html. Accessed 27 Sept. 2018.

“Western Blot.” The Human Protein Atlas, www.proteinatlas.org/learn/method/western+blot. Accessed 27 Sept. 2018.

“Western Blot.” Scitable by Nature Education, www.nature.com/scitable/topic/proteins-and-gene-expression-14122688. Accessed 27 Sept. 2018.

“Western Blotting Fundamental Principle: How Western Blot Works.” Boster, www.bosterbio.com/protocol-and-troubleshooting/western-blot-principle. Accessed 27 Sept. 2018.